PROJECT SUMMARY Bone fragility fractures are a major concern of health care of our rapidly aging populations due to the high risk of long-term disability and even premature mortality. Such fractures are not only due to loss of bone mineral density (BMD), but also due to adverse composition/structural changes at different hierarchies of bone. It is a well-known fact that bone loses its toughness completely when dehydrated. However, the underlying mechanism is still elusive. Our preliminary results suggest that proteoglycans (PGs), a sub group of non-collagenous proteins (NCPs) in bone matrix, play a pivotal role in bone tissue toughness. In addition, our results also reveal that PGs in bone matrix decreases with aging with the associated deterioration of bone toughness. Moreover, our pilot study shows that accumulated non-enzymatic glycation decreases PGs and this decrease can be compensated by delivering GAGs to bone matrix by subdermal administration, thus improving the toughness of bone. To this end, we hypothesize that (1) PGs contain glycosaminoglycans (GAGs) that attract and retain bound water in bone matrix, thus regulating the in situ hydration status of bone matrix and subsequently imposing a significant effect on the toughness of bone. (2) Aging may cause loss of GAGs/PGs in bone matrix, thus leading to significant deterioration of bone toughness, whereas supplement of GAGs may deter such age-related deterioration of bone toughness. We proposed two specific aims to address the hypotheses. Aim 1: Determine the underlying mechanism of GAGs/PGs in toughening of bone. Here, we will use in vitro human cadaveric bone, in vivo mouse, and computational models to test the hypothesis in three subaims: (1) Determine the role of GAGs in retaining bound water in bone matrix using an in vitro model. (2) Determine the role of GAGs/PGs in toughening of bone in vivo using KO mouse models. (3) Verify the mechanistic role of GAGs in toughening bone using a computational approach. Aim 2: Determine the age-related loss in GAGs/PGs and its contribution to the age-related deterioration of bone toughness. Here, we will use ex vivo human cadaver bone and in vivo animal models to test the hypothesis in three subaims: (1) Determine age-related effect of GAGs/PGs on the toughness of cortical and trabecular bone for both genders using human cadaveric bone samples. (2) Determine the effect of nonenzymatic glycation on the synthesis of PGs by bone cells using a mouse bone ex vivo model and osteoblast cell models. (3) Determine the efficacy of supplemental GAGs in deterring age-related loss of GAGs and maintaining the toughness of bone using an aging rat model. Upon completion of this aim, we expect to understand the mechanistic role of GAGs/PGs in the age and gender-related deterioration of bone toughness, a potential pathway of age-related loss of GAGs, and the efficacy of supplementing GAGs in deterring age-related loss of bone toughness. The outcomes of this study will provide important insights to age-related bone fragility fractures and facilitate development of new strategies in prediction and prevention of fragility fractures in aged population.